The AXL (Ark, UFO, Tyro-7) receptor tyrosine kinase is a member of the Tyro-3 family of kinases with the other members being Mer (Eyk, Nyk, Tyro-12) and Sky (Rse, Tyro-3, Dtk, Etk, Brt, Tif). It is activated by binding of the heterophilic ligand Gas6, a 70-kDa protein homologous to the anti-coagulation factor protein S. In contrast to other receptor tyrosine kinases, AXL tyrosine phosphorylation can also be induced by homophilic binding. AXL activation leads to signalling through PI-3-kinase/Akt (Franke et al., Oncogene 22: 8983-8998, 2003) and other major pathways like Ras/Erk and β-catening/TCF (Goruppi et al., Mol. Cell. Biol. 21: 902-915, 2001).
AXL is weakly expressed in a range of normal tissues, including brain, heart, skeletal muscle, the organ capsules and connective tissues of several other organs, and in monocytes, but not lymphocytes. Akt phosphorylation induced by AXL has been described in survival of fibroblasts (Goruppi et al., Mol Cell Biol 17: 4442-4453 1997), endothelial cells (Hasanbasic et al., Am J Physiol Heart Circ Physiol, 2004), vascular smooth muscle cells (Melaragno et al., J. Mol. Cell. Cardiol. 37: 881-887, 2004) and neurons (Allen et al., Mol. Endocrinol. 13: 191-201 1999). Furthermore, AXL plays a role in cell-adhesion and chemotaxis. AXL knockouts display impaired platelet aggregate stabilization and thrombus formation as a result of reduced activation of the platelet integrin IIb3.
AXL overexpression has been demonstrated in various cancer types, e.g. breast (Meric et al., Clin. Cancer Res. 8: 361-367, 2002; Berclaz et al., Ann. Oncol. 12: 819-824, 2001), colon (Chen et al., Int. J. Cancer 83: 579-584, 1999; Craven et al., Int. J. Cancer 60: 791-797, 1995), prostate (Jacob et al., Cancer Detect. Prey. 23: 325-332, 1999), lung (Wimmel et al., Eur J Cancer 37: 2264-2274, 2001), gastric (Wu et al., Anticancer Res 22: 1071-1078, 2002), ovarian (Sun et al., Oncology 66: 450-457, 2004), endometrial (Sun et al., Ann. Oncol. 14: 898-906, 2003), renal (Chung et al., DNA Cell Biol. 22: 533-540, 2003), hepatocellular (Tsou et al., Genomics 50:331-340, 1998), thyroid (Ito et al., Thyroid 12:971-975, 2002; Ito et al., Thyroid 9: 563-567, 1999), and esophageal carcinoma (Nemoto et al., 1997), furthermore in CML (Janssen et al., A novel putative tyrosine kinase receptor with oncogenic potential. Oncogene, 6: 2113-2120, 1991; Braunger et al., Oncogene 14:2619-2631 1997; O'Bryan et al., Mol Cell Biol 11:5016-5031, 1991), AML (Rochlitz et al., Leukemia 13: 1352-1358, 1999), osteosarcoma (Nakano et al., J. Biol. Chem. 270:5702-5705, 2003) melanoma (van Ginkel et al., Cancer Res 64:128-134, 2004) and in head and neck squamous cell carcinoma (Green et al., Br J. Cancer. 2006 94:1446-5, 2006).
Moreover AXL has been identified as a metastasis-associated gene that is upregulated in aggressive breast cancer cell lines compared to non-invasive cells. In vitro, AXL activity was found to be required for migration and invasion, and this activity could be inhibited by antibody treatment (WO04008147). Similarly, abrogation of AXL activity in vivo, either via expression of a dominant negative version of AXL (Vajkoczy, P., et al., Proc. Natl. Acad. Science U.S.A. 103: 5799-5804. 2005) or by siRNA mediated downregulation of AXL (Holland et al., Cancer Res. 65: 9294-9303, 2005) prevented subcutaneous and orthotopic cell growth in murine xenograft experiments.
So far antibodies that bind to AXL and posses biological activity have been described. For example, one known antibody is capable of reducing AXL mediated cell invasion (WO04008147) whereas an other antibody has been reported to reduce AXL/Ligand interaction. However, all known antibodies are polyclonal or non-humanized monoclonal antibodies. Non-humanized antibodies, polyclonal or monoclonal, are rapidly removed from circulation and usually cause systemic inflammatory effects, rendering them unsuitable for therapeutic administration.
Thus in light of the therapeutic potential of AXL there is a high need for monoclonal humanized AXL antibodies, antibody fragments or derivatives thereof that effectively and specifically block AXL mediated signal transduction and which are suitable for therapeutic treatment.
Accordingly a first aspect of the present invention relates to a monoclonal humanized antibody including a fragment or derivative thereof that binds to the extracellular domain of AXL, particularly of human AXL, and at least partially inhibits AXL activity.
Preferably the humanized antibody of the present invention possesses at least one or more of the following properties: the ability to reduce or block AXL-mediated signal transduction, the ability to reduce or block AXL phosphorylation, the ability to reduce or block cell proliferation, the ability to reduce or block angiogenesis, the ability to reduce or block cell migration, the ability to reduce or block tumor metastasis, and the ability to reduce or block AXL mediated anti-apoptosis, thereby increasing for example the sensitivity of a cell against treatment with an antineoplastic agent.
According to an especially preferred embodiment of the invention the humanized antibodies described herein show the ability to reduce and/or block ligand induced phosphorylation of AXL downstream signaling molecules such as ERK1/2, AKT, GSK-3β, TSC2, mTOR and/or S6K1.
Moreover the humanized antibodies of the present invention may exhibit high specificity for AXL, particularly human AXL and do not significantly recognize other Tyro-3 family members, e.g. MER and/or SKY and/or mammalian non-primate AXL, such as murine AXL.
The term “activity” as used herein refers to the biological function of AXL, which influences the phenotype of a cell, in particular but not limited to cancer phenotypes such as evasion of apoptosis, self sufficiency in growth signals, cell proliferation, tissue invasion and/or metastasis, insensitivity to anti-growth signals (anti-apoptosis) and/or sustained angiogenesis.
The term “AXL mediated signal transduction” means the activation of second messenger pathways, such as downstream signaling, triggered by direct or indirect interaction of AXL with second messenger molecules.
The term “AXL phosphorylation” refers to the phosphorylation of amino acid residues, preferably tyrosine residues, either by a second AXL protein (transphosphorylation) or by another protein having protein kinase activity.
The term “cell proliferation” refers to all AXL-involving processes underlying the reproduction of human cells, in particular but not limited to human cancer cells. They contribute to or result in the replication of cellular DNA, separation of the duplicated DNA into two equally sized groups of chromosomes, and the physical division (called cytokinesis) of entire cells, and shall be stimulated or mediated by non-catalytic or catalytic activities of AXL, preferably including AXL phosphorylation and/or AXL-mediated signal transduction.
The term “angiogenesis” refers to all AXL-involving processes that contribute to the growth of new blood vessels from pre-existing vessels, in particular but not limited to new tumor supplying blood vessels. These processes include multiple cellular events such as proliferation, survival, migration and sprouting of vascular endothelial cells, attraction and migration of pericytes as well as basal membrane formation for vessel stabilization, vessel perfusion, or secretion of angiogenic factors by stromal or neoplastic cells, and shall be stimulated or mediated by non-catalytic or catalytic activities of AXL, preferably including AXL phosphorylation and/or AXL-mediated signal transduction.
The term “metastasis” refers to all AXL-involving processes that support cancer cells to disperse from a primary tumor, penetrate into lymphatic and/or blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasis) in normal tissues elsewhere in the body. In particular, it refers to cellular events of tumor cells such as proliferation, migration, anchorage independence, evasion of apoptosis, or secretion of angiogenic factors, that underly metastasis and are stimulated or mediated by non-catalytic or catalytic activities of AXL, preferably including AXL phosphorylation and/or AXL-mediated signal transduction.
The term “AXL mediated anti-apoptosis” refers to all AXL-involving processes that prevent human cells, preferably but not limited to human cancer cells from programmed cell death (apoptosis). In particular, it refers to processes that prevent human cells, preferably but not limited to human cancer cells from induction of apoptosis through growth factor withdrawal, hypoxia, exposure to chemotherapeutic agents or radiation, or initiation of the Fas/Apo-1 receptor-mediated signaling, and are stimulated or mediated by non-catalytic or catalytic activities of AXL, preferably including AXL phosphorylation and/or AXL-mediated signal transduction.
The terms “region” and “domain” are compatible with each other in the invention.
The antibodies designated as “11B7” and “11D5” may be also designated as “#11B7” and “#11D5”, respectively, in the invention.
According to a second aspect of the invention the humanized antibodies described herein are derived from one of the chimeric (rat/human) anti-AXL antibodies 11B7 (the amino acid sequence of its light and heavy chain are represented by SEQ ID NO: 135 and 136, respectively), 11D5 (the amino acid sequence of its light and heavy chain are represented by SEQ ID NO: 137 and 138, respectively) or 10D12 or an antibody recognizing the same epitope on the extracellular domain of AXL. Particular preferred, the humanized antibodies are derived from 11B7 and 11D5. Preferably the humanized antibody contains at least one mutation in the frame work region of at least one variable domain. Such mutations may be introduced by any method known to the person skilled in the art to alter amino acid sequences. The mutation replaces preferably an amino acid in an frame work region of 11B7 or 11D5 by an amino acid which is conserved in human frame work regions. Methods for determining conserved or consensus amino acids in human frame work regions, such for example homology modelling using programs such as IgBLAST are known to the person skilled in the art. According to another preferred embodiment, the humanized antibodies of the invention are derived from anti-AXL antibodies, preferably from anti-AXL antibodies 11B7 or 11D5, wherein the frame work regions of at least one variable domain of the antibody, preferably 11B7 or 11D5, been replaced by a human or humanized frame work region.
The binding activity of an antibody of the present invention to AXL can be determined by methods known to those skilled in the art. For example, the activity can be determined using surface plasmon resonance with Biacore, and/or by ELISA (enzyme-linked immunosorbent assays), EIA (enzyme immunoassays), RIA (radioimmunoassays), or fluorescent antibody techniques, e.g. FACS.
Preferably the humanized antibodies of the invention are less immunogenic if compared with polyclonal or monoclonal non-humanized anti-AXL antibodies such as rat 11B7 or 11D5. Beside other methods known to those skilled in the art reduced immunogenicity may be determined by ELISA based HAHA or HAMA assays (IBL-America, Minnesota/LiSrarFish, Italy). Moreover, preferably the inventive antibodies are not rapidly removed from blood circulation and do not cause systemic inflammatory effects if administered to a patient.
The antibodies of the invention may have at least one antigen binding site, e.g. one or two antigen binding sites. Further, the antibody preferably comprises at least one heavy immunoglobulin chain and at least one light immunoglobulin chain. An immunoglobulin chain comprises a variable domain and optionally a constant domain. A variable domain may comprise complementary determining regions (CDRs), e.g. a CDR1, CDR2 and/or CDR3 region, and framework regions. The term “complementary determining region” (CDR) is well-defined in the art (see, for example, Harlow and Lane, “Antibodies, a Laboratory Manual”, CSH Press, Cold Spring Harbour, 1988) and refers to the stretches of amino acids within the variable region of an antibody that primarily makes contact with the antigen.
According to a further embodiment the humanized antibodies of the invention are derived from the anti-AXL antibody 11B7 and may comprise a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:40 to SEQ ID NO:48, or at least the variable domain thereof, or an amino acid sequence having a sequence identity of at least 90% thereto and represents a variable region having binding activity to AxI which is equivalent to that of one having a sequence identity of 100%    or a variable domain of a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:80 to SEQ ID NO:82 or an amino acid sequence having a sequence identity of at least 90% thereto and represents a variable region having binding activity to AxI which is equivalent to that of one having a sequence identity of 100%    and/or a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO:30, or at least the variable domain thereof or an amino acid sequence having a sequence identity of at least 90% thereto and represents a variable region having binding activity to Axl which is equivalent to that of one having a sequence identity of 100%,    or a variable domain of a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 73 to SEQ ID NO:79 or an amino acid sequence having a sequence identity of at least 90% thereto and represents a variable region having binding activity to Axl which is equivalent to that of one having a sequence identity of 100%,    or a fragment thereof recognizing the same epitope on the extracellular domain of AXL. A description of the sequences according to the invention is presented below under “Description of the Sequences” and in the Sequence Listing.
According to another further embodiment the humanized antibodies of the invention are derived from the anti-AXL antibody 11B7 and may comprise a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:150 and SEQ ID NO:151, or at least the variable domain thereof, or an amino acid sequence having a sequence identity of at least 90% thereto and represents a variable region having binding activity to Axl which is equivalent to that of one having a sequence identity of 100%,    and/or a light chain amino acid sequence selected from the group consisting of SEQ ID NO: 146 and SEQ ID NO:147, or at least the variable domain thereof or an amino acid sequence having a sequence identity of at least 90% thereto and represents a variable region having binding activity to Axl which is equivalent to that of one having a sequence identity of 100%.
In particular preferred embodiment the humanized h#11B7 antibodies are selected from the group consisting of h#11B7-T1, h#11B7-T2, h#11B7-T3, h#11B7-T4, h#11B7-T5, h#11B7-T6, h#11B7-T7, h#11B7-T8, h#11B7-T9, h#11B7-T10, h#11B7-T11, h#11B7-T12, h#11B7-T13, h#11B7-T14, h#11B7-T15, h#11B7-T16, h#11B17-T17, h#11B7-T18, h#11B7-T19, h#11B7-T20, h#11B7-T21, h#11B7-T22, h#11B7-T23, h#11B7-T24, h#11B7-T25, h#11B7-T26, and h#11B7-T27 antibodies. Table 1 (Example 10) summarizes by which combinations of specific humanized heavy and light chain amino acid sequences, respectively by which combination of corresponding expression vectors, the above humanized h#11B7 antibodies are characterized in the invention.
In another particular preferred embodiment the humanized h#11B7 antibodies are selected from the group consisting of h#11B7-T28, h#11B7-T29, h#11B7-T30 and h#11B7-T31 antibodies. Table 2 (Example 22) summarizes by which combinations of specific humanized heavy and light chain amino acid sequences, respectively by which combination of corresponding expression vectors, the above humanized h#11B7 antibodies are characterized.
A humanized antibody derived from rat anti-human Axl monoclonal antibody #11B7 is not limited to the specific antibodies exemplified in the previous paragraphs, as far as said humanized antibody retains all of six CDRs and binding activity to the Axl antigen. Said humanized antibody retains CDRH1 (SNYWG; SEQ ID NO: 124), CDRH2 (YITYSGSTSYNPSLKS; SEQ ID NO: 125) and CDRH3 (TTFYY; SEQ ID NO: 126) in its heavy chain, and, CDRL4 (RASQDIGNYLR; SEQ ID NO: 121), CDRL5 (GATNLAA; SEQ ID NO: 122) and CDRL6 (LQSKESPWT; SEQ ID NO:123) in its light chain, respectively.
According to a further embodiment the humanized antibodies of the invention are derived from 11D5 and comprise a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:67 to SEQ ID NO:72, or at least the variable domain thereof or an amino acid sequence having a sequence identity of at least 90% thereto and represents a variable region having binding activity to Axl which is equivalent to that of one having a sequence identity of 100%,    or a variable domain of a heavy chain amino acid sequence selected from the group consisting of SEQ ID NO:114 to SEQ ID NO:120 or an amino acid sequence having a sequence identity of at least 90% thereto and represents a variable region having binding activity to Axl which is equivalent to that of one having a sequence identity of 100%    and/or a light chain amino acid sequence selected from the group consisting of SEQ ID NO:55 to SEQ ID NO:60, or at least the variable domain thereof or an amino acid sequence having a sequence identity of at least 90% thereto and represents a variable region having binding activity to Axl which is equivalent to that of one having a sequence identity of 100%,    or a variable domain of a light chain amino acid sequence selected from the group consisting of SEQ ID NO:83 to SEQ ID NO:113    or an amino acid sequence having a sequence identity of at least 90% thereto and represents a variable region having binding activity to Axl which is equivalent to that of one having a sequence identity of 100%, or a fragment thereof recognizing the same epitope on the extracellular domain of AXL.
In particular preferred embodiment the humanized h#11D5 antibodies are selected from the group consisting of h#11D5-T1, h#11D5-T2, h#11D5-T3, h#11D5-T4, h#11D5-T5 or h#11D5-T6 antibodies. Examples 11, 12 and 14 show by which combinations of specific humanized heavy and light chain amino acid sequences, respectively by which combination of corresponding expression vectors, the above humanized h#11D5 antibodies are characterized.
One of the preferred embodiments of the present invention is a humanized antibody having a relatively high thermal denaturation midpoint (Tm). In this invention, a humanized antibody has a Tm of at least 70° C., preferably, a Tm of at least 75° C., more preferably, a Tm of at least 78° C., moreover preferably, a Tm of at least 80° C., further moreover preferably, 82° C. Or, in the present invention, a humanized antibody has a Tm equivalent to the Tm of its ancenstor rat or chimeric antibody, but, preferably, a Tm of at least 3° C. higher, more preferably, a Tm of at least 6° C. higher, moreover preferably, a Tm of at least 8° C. higher, further moreover preferably, a Tm of at least 10° C. higher, than the Tm of its ancestor rat or chimeric antibody. Such a preferred, more preferred, moreover preferred or further moreover preferred humanized antibody is less prone to be denatured (unfolded) or inactivated, and is suitable to be prepared into solution formulations that can be stored stably for a long time.
A humanized antibody derived from rat anti-human Axl monoclonal antibody #11D5 is not limited to the specific antibodies exemplified in the previous paragraphs, as far as said humanized antibody retains all of six CDRs and binding activity to the Axl antigen. Said humanized antibody retains CDRH1 (SNYWG, SEQ ID NO: 130), CDRH2 (HITNSGNTTYNPSLKS; SEQ ID NO: 131) and CDRH3 (GAFDY; SEQ ID NO: 132) in its heavy chain, and, CDRL4 (RASQDIGNYLS; SEQ ID NO: 127), CDRL5 (GAIKLAV; SEQ ID NO: 128) and CDRL6 (LQYIQFPLT; SEQ ID NO: 129) in its light chain, respectively.
Another preferred embodiment of the invention is an antibody which (specifically) binds or recognizes one of IG domains closer to the carboxy terminus (the domain comprising amino acid residues of amino acid Nos. 129-220 in NCBI protein database ACCESSION No. P—30530: SEQ ID NO: 139; FIG. 30A). Such a preferred antibody is not limited to a humanized antibody, but can be a human antibody or a functional fragment of one of them. More preferably, such a preferred antibody inhibits at least one of the biological activities that Axl has.
A (full) antibody of the present invention can be produced, for example, by carrying out the following steps: constructing a heavy chain expression vector and light chain expression vector, wherein each of said vector has an insert comprising a variable region and constant region; introducing said vectors into a host cell; culturing said cell; recovering antibody polypeptides from the culture supernatant (the conditioned medium), as exemplified in Examples 9 to 12.
As used herein, “sequence identity” between two polypeptide sequences, indicates the percentage of amino acids that are identical between the sequences. Methods how to determine the “sequence identity” between two given polypeptide sequences are known to the person skilled in the art. Preferred polypeptide sequences of the invention have a sequence identity of at least 90%, more preferably of al least 95% and even more preferably of at least 98%. The sequence identity can be determined, for example, by BLAST, an algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 90, pp 5873 (1993)) or FASTA (Methods in Enzymol., 183, pp 63 (1990)). A program designated BLASTN or BLASTX is available (J. Mol. Biol., 215, pp 403 (1990)).
The antibody of the invention may be of the IgA-, IgD-, IgE, IgG- or IgM-type, preferably of the IgG- or IgM-type including, but not limited to, the IgG2-, IgG3-, IgM1- and IgM2-type.
Inventive antibodies may be preferably designed by homology modeling using any suitable procedure known to the person skilled in the art Moreover, humanized forms of given antibodies, such as 11B7 or 11D5, may be generated according to the methods known in the art such as chimerization or CDR grafting. Alternative methods for the production of humanized antibodies are well known in the art and are described in, e.g., EP-A1 0 239 400 and WO9007861. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be for example performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting CDRs or CDR sequences of non human origin for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in non human antibodies.
A human antibody can be derived from a phage-display library of human antibodies (Wormstone, I. M. et. al, Investigative Ophthalmology & Visual Science. (2002) 43 (7), p. 2301-2308; Carmen, S. et. al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p. 189-203; Siriwardena, D. et. al., Opthalmology (2002) 109 (3), p. 427-431), which is well-known to the skilled person.
For example, a phage display method, which comprises having a variable region of human antibody expressing on the surface of phage as a single chain antibody, scFv, and selecting a phage which binds to an antigen, can be used (Nature Biotechnology (2005), 23, (9), p. 1105-1116).
By analyzing the gene of the selected phage, the DNA sequence encoding the variable region of a human antibody binding to the antigen can be determined.
After the DNA sequence of the scFv is determined, a human antibody can be produced by constructing an expression vector comprising said sequence and introducing said vector into a suitable host cell to express said antibody (WO92001047, WO92020791, WO93006213, WO93011236, WO93019172, WO95001438, WO95015388 and Annu. Rev. Immunol (1994) 12, p. 433-455 and Nature Biotechnology (2005) 23 (9), p. 1105-1116).
Any human antibody, regardless of a method for the production thereof, can be an embodiment of the present invention, as far as said human antibody has binding activity to human Axl antigen, and all of the following six CDRs: CDRH1 (SNYWG; SEQ ID NO: 130), CDRH2 (HITNSGNTTYNPSLKS; SEQ ID NO: 131) and CDRH3 (GAFDY; SEQ ID NO: 132) in its heavy chain, and, CDRL4 (RASQDIGNYLS; SEQ ID NO: 127), CDRL5 (GAIKLAV; SEQ ID NO: 128) and CDRL6 (LQYIQFPLT; SEQ ID NO: 129) in its light chain, respectively, alternatively, as far as said human antibody has binding activity to human Axl antigen, and all of the following six CRDs: CDRH1 (SNYWG; SEQ ID NO: 124), CDRH2 (YITYSGSTSYNPSLKS; SEQ ID NO: 125) and CDRH3 (TTFYY; SEQ ID NO: 126) in its heavy chain, and, CDRL4 (RASQDIGNYLR; SEQ ID NO: 121), CDRL5 (GATNLAA; SEQ ID NO: 122) and CDRL6 (LQSKESPWT; SEQ ID NO:123) in its light chain, respectively.
For therapeutic purposes, the antibody may be conjugated with a therapeutic effector group, e.g. a radioactive group or a cytotoxic group.
For diagnostic purposes, the antibody may be labeled. Suitable labels include radioactive labels, fluorescent labels, or enzyme labels.
As discussed above, the antibody of the invention may exist in a variety of forms besides complete antibodies; including, for example, Fv, Fab, Fab′ and F(ab′)2, diabody, minibody, divalent or multivalent antibody comprising a fragment of more than one antibody, as well as in single chains (scFV); see e.g. WO8809344.
An antibody of the present invention can be a bi-specific antibody or mutli-specific antibody which is specific for more than one antigen or epitope.
A scFv can be obtained by fusing a variable region of an immunoglobulin heavy chain to the variable region of an immunoglobulin light chain through a polypeptide linker (Pluckthun, The Pharmacology of Monoclonal Antibodies, 113, edited by Rosenburg and Moore, Springer Verlag, New York, p. 269-315 (1994) and Nature Biotechnology (2005), 23, p. 1126-1136). A BiscFv can be obtained by fusing two different scFV through a polypeptide linker.
Methods for generating scFv are well-know to the person skilled in the art (U.S. Pat. Nos. 4,946,778, 5,260,203, 5,091,513, 5,455,030 etc). Preferably, a linker between two variable regions does not produce a conjugate (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, p. 5879-5883). A scFV immunoglobulin heavy chain variable region and a scFV immunoglobulin light chain variable region can be derived from an identical antibody or two antibodies, one of which is different from the other. A polypeptide linker can be a single chain peptide consisting of, for example, 12 to 19 amino acid residues.
A DNA encoding a scFV can be generated by PCR which comprises amplifying a DNA fragment using a DNA encoding a full length or portion of an immunoglobulin heavy chain variable region or light chain variable region and two oligonucleotides corresponding to each of both terminuses as a template as a pair of primers, respectively, and then amplifying using a pair of primers which are designed so that a heavy chain variable region and light chain variable region are connected to one terminus and the other terminus of a polypeptide linker, respectively.
A functional fragment of an antibody including scFv can be produced by introducing a DNA encoding said scFv into a host cell and culturing said cell, as described elsewhere in the description.
One of the embodiments of the invention is a multimeric antibody that has higher binding affinity to an antigen than a monomeric antibody. The Unit of said multimeric antibody can be the same, or different from each other when a unit binds to an epitope of an antigen and another unit binds to a different epitope of the same antigen. Binding of IgG CH3 domain and two scFv, binding with streptoavidin and introduction of a helix-turn-helix motif can be applied for producing multimeric antibody.
If desired, the antibodies of the invention may be mutated in the variable domains of the heavy and/or light chains to alter a binding property of the antibody. For example, a mutation may be made in one or more of the CDR regions to increase or decrease the Kd of the antibody for AXL, or to alter the binding specificity of the antibody. Techniques in site directed mutagenesis are well-known in the art. See, e.g., Sambrook et al. and Ausubel et al., supra. Furthermore, mutations may be made at an amino acid residue that is known to be changed compared to germline in a variable region of an AXL antibody.
In another aspect, beside mutations with respect to humanization, further mutations may be introduced into one or more of the framework regions. A mutation may be made in a framework region or constant domain to increase the half-life of the AXL antibody. See, e.g., WO0009560. A mutation in a framework region or constant domain may also be made to alter the immunogenicity of the antibody, to provide a site for covalent or non-covalent binding to another molecule, or to alter such properties as complement fixation. Mutations may be made in each of the framework regions, the constant domain and the variable regions in a single mutated antibody. Alternatively, mutations may be made in only one of the framework regions, the variable regions or the constant domain in a single mutated antibody.
One of the embodiments of the invention is a polyclonal antibody comprising more than one antibody of the present invention.
One of the embodiments of the invention is a chemical modification of an antibody or its functional fragment of the present invention. Molecules such as polymer (polyethylene glycol or the like) can be used to generate a modified antibody or modified functional fragment.
In a further aspect, the humanized antibody according to the invention may have a constant domain with effector functions, whereby AXL expressing cells which have bound the antibody, antibody fragment or derivative thereof on the cell surface may be attacked by immune system functions. For example, the antibody may be capable of fixing complement and participating in complement-dependent cytotoxicity (CDC). Moreover, the antibody may be capable of binding to Fc receptors on effector cells, such as monocytes and natural killer (NK) cells, and participate in antibody-dependent cellular cytotoxicity (ADCC).
In yet a further aspect the antibodies described herein are applicable for therapeutic treatment, preferably for treatment of hyperproliferative diseases, cardiovascular diseases, in particular artherosclerosis and thrombosis, diabetes related diseases, in particular glomerular hypertrophy or diabetic nephropathy, and particularly of disorders associated with, accompanied by or caused by AXL expression, overexpression or hyperactivity. The hyperproliferative diseases are preferably selected from disorders associated with, accompanied by or caused by AXL expression, overexpression or hyperactivity, such as cancer, e.g. breast cancer, colon cancer, lung cancer, kidney cancer, follicular lymphoma, myeloid leukemia, skin cancer/melanoma, glioblastoma, ovarian cancer, prostate cancer, pancreatic cancer, Barrett's esophagus and esophageal cancer, stomach cancer, bladder cancer, cervical cancer, liver cancer, thyroid cancer, and head and neck cancer, or hyperplastic and neoplastic diseases or other AXL expressing or overexpressing hyperproliferative diseases.
The applicability of the antibodies of the present invention for the treatment of any Axl-related diseases such as hyperproliferative diseases can be demonstrated, indicated or suggested by in vitro, in vivo or ex vivo experiments, including those on ligand-induced autophosphorylation of Axl, effect on Axl-mediated downstream signal trancduction such as ligand-induced Akt and p42/44 MAP-kinase phosphorylation, cancer cell migration or proliferation, ligand-induced migration or proliferation of a cell expressing Axl, angiogenesis of tissues or cells, and growth or metastasis of human cancer or cancer cell transplanted onto non-human animals such as xenograft mice, as well as any other pre-clinical/non-clinical experiments and clinical trials.
In another aspect the antibodies of the present invention can be used for the co-administration with an antineoplastic agent for the treatment of one of the above mentioned disorders.
Co-administration as used herein includes the administration of an antibody of the present invention with an antineoplastic agent, preferably an apoptosis inducing antineoplastic agent. The term co-administration further includes the administration of the antibody of the present invention and the antineoplastic agent, preferably an apoptosis inducing antineoplastic agent, in the form of a single composition or in the form of two or more distinct compositions. Co-administration includes the administration of an antibody of the present invention with an antineoplastic agent, preferably an apoptosis inducing antineoplastic agent simultaneously (i.e. at the same time) or sequentially, (i.e. at intervals).
The invention further relates to a nucleic acid molecule encoding the antibody, antibody fragment or derivative thereof of the invention. The nucleic acid molecule of the invention encoding the above-described antibody, antibody fragment or derivative thereof may be, e.g. DNA, cDNA, RNA or synthetically produced DNA or RNA or recombinantly produced chimeric nucleic acid molecule comprising any of those nucleic acid molecules either alone or in combination. The nucleic acid molecule may also be genomic DNA corresponding to the entire gene or a substantial portion thereof or to fragments and derivatives thereof. The nucleotide sequence may correspond to the naturally occurring nucleotide sequence or may contain single or multiple nucleotide substitutions, deletions or additions. In a particular preferred embodiment of the present invention, the nucleic acid molecule is a cDNA molecule.
According to a further aspect, the present invention relates to an isolated nucleic acid molecule selected from the group consisting of:                (a) a nucleic acid sequence encoding a monoclonal antibody according to the present invention, antibody fragment or a derivative thereof,        (b) a nucleic acid sequence as shown in one of the SEQ ID NOs. selected from the group consisting of SEQ ID NO:5 to SEQ ID NO:17, SEQ ID NO:31 to SEQ ID NO:39, SEQ ID NO:49 to SEQ ID NO:54 and SEQ ID NO:60 to SEQ ID NO:66        (c) a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NO:18 to SEQ ID NO:30, SEQ ID NO:40 to SEQ ID NO:48, SEQ ID NO:55 to SEQ ID NO:60, SEQ ID NO:67 to SEQ ID NO:72, SEQ ID NO:73 to SEQ ID NO:120 and SEQ ID NO:121 to 132,        (d) a nucleic acid complementary to any of the sequences in (a) to (c), or        (e) a nucleic acid sequence capable of hybridizing to (a), (b), (c) or (d) under stringent conditions and encoding a polypeptide, wherein an antibody or functional fragment thereof comprising said polypeptide binds to the extracellular domain of AXL,        (f) a nucleic acid sequence as shown in one of the SEQ ID NOs. selected from the group consisting of SEQ ID NO:144 and SEQ ID 145 and SEQ ID NO:148 and SEQ ID NO:149,        (g) a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ ID NO: 146 and SEQ ID NO: 147 and SEQ ID NO: 150 and SEQ ID NO:151,        (h) a nucleic acid complementary to any of the sequences in (f) or (g), or        (i) a nucleic acid sequence capable of hybridizing to (f), (g), or (h) under stringent conditions, and encoding a polypeptide, wherein antibody or functional fragment thereof comprising said polypeptide binds to the extracellular domain of AXL.        
The term “hybridizing under stringent conditions” means that two nucleic acid fragments hybridize with one another under standardized hybridization conditions as described for example in Sambrook et al., “Expression of cloned genes in E. coli” in Molecular Cloning: A laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, USA. Such conditions are for example hybridization in 6.0×SSC at about 45° C. followed by a washing step with 2.0×SSC at 50° C., preferably 2.0×SSC at 65° C., or 0.2×SSC at 50° C., preferably 0.2×SSC at 65° C.
The invention also relates to a vector comprising a nucleic acid molecule of the invention. Said vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
The nucleic acid molecules of the invention may be joined to a vector containing selectable markers for propagation in a host. Generally, a plasmid vector is introduced in a precipitate such as a calcium phosphate precipitate or rubidium chloride precipitate, or in a complex with a charged lipid or in carbon-based clusters, such as fullerens. Should the vector be a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells.
Preferably, the vector of the invention is an expression vector wherein the nucleic acid molecule is operatively linked to one or more control sequences allowing the transcription and optionally expression in prokaryotic and/or eukaryotic host cells. Expression of said nucleic acid molecule comprises transcription of the nucleic acid molecule, preferably into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the lac, tip or tac promoter in E. coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOXI or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (Invitrogen) or pSPORTI (GIBCO BRL). Preferably, said vector is an expression vector and/or a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector of the invention into targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (2001, Third Edition) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994). Alternatively, the nucleic acid molecules of the invention can be reconstituted into liposomes for delivery to target cells.
The invention further relates to a host comprising the vector of the invention. Said host may be a prokaryotic or eukaryotic cell or a non-human transgenic animal. The polynucleotide or vector of the invention which is present in the host may either be integrated into the genome of the host or it may be maintained extrachromosomally. In this respect, it is also to be understood that the nucleic acid molecule of the invention can be used for “gene targeting” and/or “gene replacement”, for restoring a mutant gene or for creating a mutant gene via homologous recombination; see for example Mouellic, Proc. Natl. Acad. Sci. USA, 87 (1990), 4712-4716; Joyner, Gene Targeting, A Practical Approach, Oxford University Press.
The host can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal, mammalian or, preferably, human cell. Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae. The term “prokaryotic” is meant to include all bacteria which can be transformed or transfected with a polynucleotide for the expression of a variant polypeptide of the invention. Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. A polynucleotide coding for a mutant form of variant polypeptides of the invention can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art. Methods for preparing fused, operably linked genes and expressing them in bacteria or animal cells are well-known in the art (Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (2001, Third Edition). The genetic constructs and methods described therein can be utilized for expression of variant antibodies, antibody fragments or derivatives thereof of the invention in, e.g., prokaryotic hosts. In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted nucleic acid molecule are used in connection with the host. The expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells. The transformed prokaryotic hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth. The antibodies, antibody fragments or derivatives thereof of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions. The isolation and purification of the microbially or otherwise expressed antibodies, antibody fragments or derivatives thereof of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies.
In a preferred embodiment of the invention, the host is a bacterium, fungal, plant, amphibian or animal cell. Preferred animal cells include but are not limited to Chinese hamster ovary (CHO) cells (CHO cell, ATCC CCL-61) or a dihydrofolate reductaase-deficient strain thereof (Urlaub, G. and Chasin, L. A. Proc. Natl. Acad. Sci. U.S.A. (1980) 77, p. 4126-4220), baby hamster kidney (BHK) cells, monkey kidney cells (COS) (Gluzman, Y. Cell (1981) 23, p. 175-182 and ATCC CRL-1650), 3T3 cells or NIH3T3 cells derived from a mouse fibroblast (ATCC No. CRL-1658), NSO cells and a number of other cell lines including human cells, for example Per.C6. In another preferred embodiment, said animal cell is an insect cell. Preferred insect cells include but are not limited to cells of the SF9 cell lines.
In a more preferred embodiment of the invention, said host is a human cell or human cell line. Said human cells include, but are not limited to Human embryonic kidney cells (HEK293, 293T, 293 freestyle). Furthermore, said human cell lines include, but are not limited to HeLa cells, human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells.
The invention also provides transgenic non-human animals comprising one or more nucleic acid molecules of the invention that may be used to produce antibodies of the invention. Antibodies can be produced in and recovered from tissue or body fluids, such as milk, blood or urine, of goats, cows, horses, pigs, rats, mice, rabbits, hamsters or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690; 5,756,687; 5,750,172; and 5,741,957. As described above, non-human transgenic animals that comprise human immunoglobulin loci can be produced by immunizing with AXL or a portion thereof.
The invention additionally relates to a method for the preparation of an antibody, comprising culturing the host of the invention under conditions that allow synthesis of said antibody and recovering said antibody from said culture.
The transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth. Once expressed, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention, can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like; see, Scopes, “Protein Purification”, Springer-Verlag, N.Y. (1982). The antibody or its corresponding immunoglobulin chain(s) of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions. The isolation and purification of the, e.g., microbially expressed antibodies or immunoglobulin chains of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies directed, e.g., against the constant region of the antibody of the invention.
It will be apparent to those skilled in the art that the antibodies of the invention can be further coupled to other moieties for, e.g., drug targeting and imaging applications. Such coupling may be conducted chemically after expression of the antibody or antigen to site of attachment or the coupling product may be engineered into the antibody or antigen of the invention at the DNA level. The DNAs are then expressed in a suitable host system, and the expressed proteins are collected and renatured, if necessary.
In a preferred embodiment of the present invention, the antibody is coupled to an effector, such as a radioisotope or a toxic chemotherapeutic agent. Preferably, these antibody conjugates are useful in targeting cells, e.g. cancer cells, expressing AXL, for elimination. The linking of antibodies/antibody fragments of the invention to radioisotopes e.g. provides advantages to tumor treatments. Unlike chemotherapy and other forms of cancer treatment, radioimmunotherapy or the administration of a radioisotope-antibody combination directly targets the cancer cells with minimal damage to surrounding normal, healthy tissue. Preferred radioisotopes include e.g. 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I.
Furthermore, the antibodies of the invention can be used to treat cancer when being conjugated with toxic chemotherapeutic drugs such as geldanamycin (Mandler et al., J. Natl. Cancer Inst., 92(19), 1549-51 (2000>> and maytansin, for example, the maytansinoid drug, DM1 (Liu et al., Proc. Natl. Acad. Sci. U.S.A. 93:8618-8623 (1996) and auristatin-E or monomethylauristatin-E (Doronina et al., Nat. Biotechnol. 21:778-784 (2003) or calicheamicit). Different linkers that release the drugs under acidic or reducing conditions or upon exposure to specific proteases are employed with this technology. The antibodies of the invention may be conjugated as described in the art.
One of the embodiments of the invention is a chemical modification of an antibody or its functional fragment of the present invention. Molecules such as polymer (polyethylene glycol or the like) can be used to generate a modified antibody or modified functional fragment.
The invention further relates to a pharmaceutical composition comprising the antibody, the nucleic acid molecule, the vector, the host of the invention or an antibody obtained by the method of the invention.
The term “composition” as employed herein comprises at least one compound of the invention. Preferably, such a composition is a pharmaceutical or a diagnostic composition.
It is preferred that said pharmaceutical composition comprises a pharmaceutically acceptable carrier and/or diluent. The herein disclosed pharmaceutical composition may be partially useful for the treatment of disorders associated with, accompanied by or caused by AXL expression, overexpression or hyperactivity, e.g. hyperproliferative diseases, cardiovascular diseases, in particular artherosclerosis and thrombosis, diabetes related diseases, in particular glomerular hypertrophy or diabetic nephropathy. Said disorders comprise, but are not limited to cancer, e.g. breast cancer, colon cancer, lung cancer, kidney cancer, follicular lymphoma, myeloid leukemia, skin cancer/melanoma, glioblastoma, ovarian cancer, prostate cancer, pancreatic cancer, Barrett's esophagus and esophageal cancer, stomach cancer, bladder cancer, cervical cancer, liver cancer, thyroid cancer, and head and neck cancer, or other hyperplastic or neoplastic diseases or other AXL expressing or overexpressing diseases.
The term “hyperactivity” herein refers to uncontrolled AXL signaling which may be caused by a lack and/or dysfunction of negative regulation. By way of example negative regulation comprises protein dephosphorylation, degradation and/or endocytosis. Moreover uncontrolled AXL signaling may be the result of genetic alterations, either somatic or germline, which result in changes of the AXL amino acid sequence.
Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site, like the brain. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Proteinaceous pharmaceutically active matter may be present in amounts between 1 μg and 100 mg/kg body weight per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it should also be in the range of 1 pg to 100 mg per kilogram of body weight per minute.
Progress can be monitored by periodic assessment. The compositions of the invention may be administered locally or systemically. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents depending on the intended use of the pharmaceutical composition.
It is particularly preferred that the pharmaceutical composition comprises at least one further active agent like, e.g. an additional antineoplastic agent, small molecule inhibitor, anti-tumor agent, chemotherapeutic agent or a combination of those agents.
The invention also relates to a pharmaceutical composition comprising an humanized anti-AXL-antibody of the invention in combination with at least one further antineoplastic agent. Said combination is effective, for example, in inhibiting abnormal cell growth.
Many antineoplastic agents are presently known in the art. In general the term includes all agents that are capable of prevention, alleviation and/or treatment of hyperproliferative disorders. In one embodiment, the antineoplastic agent is selected from the group of therapeutic proteins including but not limited to antibodies or immunomodulatory proteins. In another embodiment the antineoplastic agent is selected from the group of small molecule inhibitors or chemotherapeutic agents consisting of mitotic inhibitors, kinase inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, histone deacetylase inhibitors, anti-survival agents, biological response modifiers, anti-hormones, e.g. anti-androgens, and antiangiogenesis agents.
Specific examples of antineoplastic agents which can be used in combination with the antibodies provided herein include, for example, gefitinib, lapatinib, sunitinib, pemetrexed, bevacisumab, cetuximab, imatinib, trastuzumab, alemtuzumab, rituximab, erlotinib, bortezomib and the like. Other specific antineoplastic agents to be used in the compositions as described and claimed herein include for example, chemotherapeutic agents such as capecitabine, daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphor-amide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. In particular preferred are such antineoplastic agents that induce apoptosis.
The invention relates also to a polyclonal antibody comprising more than one antibody of the present invention.
When used with the described AXL antibodies, such antineoplastic agents may be used individually (e.g., 5-FU and an antibody), sequentially (e.g., 5-EU and an antibody for a period of time followed by MTX and an antibody), or in combination with one or more other such antineoplastic agents (e.g., 5-FU, MTX and an antibody, or 5-FU, radiotherapy and an antibody).
The term “antineoplastic agent” may also include therapeutic procedures, as for example irradiation or radiotherapy.
The pharmaceutical composition of the invention are preferably used in human medicine but may be used also for veterinary purposes.
Additionally, the invention relates to the use of the antibody of the invention, the nucleic acid molecule, the vector, the host of the invention or an antibody obtained by the method of the invention for the preparation of a pharmaceutical composition for diagnosis, prevention or treatment of hyperproliferative diseases, cardiovascular diseases, in particular artherosclerosis and thrombosis; diabetes related diseases, in particular glomerular hypertrophy or diabetic nephropathy, and particularly of disorders associated with, accompanied by or caused by AXL expression, overexpression or hyperactivity.
A hyperproliferative disease as mentioned above includes any neoplasia, i.e. any abnormal and/or uncontrolled new growth of tissue. The term “uncontrolled new growth of tissue” as used herein may depend upon a dysfunction and/or loss of growth regulation. A hyperproliferative disease includes tumor diseases and/or cancer, such as metastatic or invasive cancers.
In a preferred embodiment of the use of the invention, said hyperproliferative disease is in particular breast cancer, colon cancer, lung cancer, kidney cancer, follicular lymphoma, myeloid leukemia, skin cancer/melanoma, glioblastoma, ovarian cancer, prostate cancer, pancreatic cancer, Barrett's esophagus and esophageal cancer, stomach cancer, bladder cancer, cervical cancer, liver cancer, thyroid cancer, and head and neck cancer, or hyperplastic or neoplastic diseases or other AXL expressing or overexpressing hyperproliferative diseases.
In yet another embodiment the present invention refers to the use of an humanized anti-AXL-antibody of the present invention for the manufacture of a medicament for the co-administration with an antineoplastic agent for the treatment of one of the above mentioned disorders.
According to a further preferred embodiment the present invention is directed to the use of an humanized anti-AXL antibody of the invention for the manufacture of a pharmaceutical composition for the treatment of drug resistant cancer.
Further the present invention relates to a diagnostic composition comprising the antibody of the invention, the nucleic acid molecule, the vector, the host of the invention or an antibody obtained by the method of the invention and optionally a pharmaceutically acceptable carrier.
The diagnostic composition of the invention is useful in the detection of an undesired expression, overexpression or hyperactivity of the mammalian AXL in different cells, tissues or another suitable sample, comprising contacting a sample with an antibody of the invention, and detecting the presence of AXL in the sample. Accordingly, the diagnostic composition of the invention may be used for assessing the onset or the disease status of a hyperproliferative disease.
Furthermore, malignant cells, such as cancer cells expressing AXL, can be targeted with the antibody of the invention. The cells which have bound the antibody of the invention might thus be attacked by immune system functions such as the complement system or by cell-mediated cytotoxicity, thereby reducing the number of or eradicating cancer cells. These considerations equally apply to the treatment of metastases and re-current tumors.
In another aspect of the present invention, the antibody of the invention is coupled to a labelling group. As already outlined above, such antibodies are particularly suitable for diagnostic applications. As used herein, the term “labelling group” refers to a detectable marker, e.g. a radiolabelled amino acid or biotinyl moieties that can be detected by marked avidin. Various methods for labelling polypeptides and glycoproteins, such as antibodies, are known in the art and may be used in performing the present invention. Examples of suitable labelling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g. 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent groups (e.g. FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g. horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g. leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
In certain aspects, it may be desirable, that the labelling groups are attached by spacer arms of various lengths to reduce potential steric hindrance.
In another embodiment the present invention relates to a method of assessing for the presence of AXL expressing cells comprising contacting the antibody of the invention with cells or a tissue suspected of carrying AXL on their/its surface. Suitable methods for detection of AXL expression in a sample may be an Enzyme-Linked Immunosorbent Assay (ELISA) or Immunohistochemistry (IHC).
An ELISA assay may be carried out in a microtiter plate format, wherein e.g. wells of a microtiter plate, are adsorbed with an AXL antibody. The wells are rinsed and treated with a blocking agent such as milk protein or albumin to prevent nonspecific adsorption of the analyte. Subsequently the wells are treated with a test sample. After rinsing away the test sample or standard, the wells are treated with a second AXL antibody that is labelled, e.g. by conjugation with biotin. After washing away excess secondary antibody, the label is detected, e.g. with avidin-conjugated horseradish peroxidase (HRP) and a suitable chromogenic substrate. The concentration of the AXL antigen in the test samples is determined by comparison with a standard curve developed from standard samples.
For IHC, paraffin-embedded tissues may be used, wherein the tissues are, e.g. first deparaffinized in xylene and then dehydrated, e.g. with ethanol and rinsed in distilled water. Antigenic epitopes masked by formalin-fixation and paraffin-embedding may be exposed by epitope unmasking, enzymatic digestion or saponin. For epitope unmasking paraffin sections may be heated in a steamer, water bath or microwave oven for 20-40 min in an epitope retrieval solution as for example 2N HCl solution (pH 1.0). In the case of an enzyme digestion, tissue sections may be incubated at 37° C. for 10-30 minutes in different enzyme solutions such as proteinase K, trypsin, pronase, pepsin etc. After rinsing away the epitope retrieval solution or excess enzyme, tissue sections are treated with a blocking buffer to prevent unspecific interactions. The primary AXL antibody is added at appropriate concentrations. Excess primary antibody is rinsed away and sections are incubated in peroxidase blocking solution for 10 min at room temperature. After another washing step, tissue sections are incubated with a secondary labeled antibody, e.g. labeled with a group that might serve as an anchor for an enzyme. Examples therefore are biotin labeled secondary antibodies that are recognized by streptavidin coupled horseradish peroxidase. Detection of the antibody/enzyme complex is achieved by incubating with a suitable chromogenic substrate.
In an additional embodiment the present invention relates to a method of blocking AXL function comprising contacting the antibody of the invention with cells, or a tissue suspected of carrying AXL on their/its surface under conditions, wherein the antibody is capable of blocking AXL function. The contacting may be in vitro or in vivo.
The invention also relates to a method of treating a hyperproliferative disease, cardiovascular diseases, in particular artherosclerosis and thrombosis, diabetes related diseases, in particular glomerular hypertrophy or diabetic nephropathy, comprising, administering to a patient in need thereof a suitable dose of the antibody or antibody fragment or derivative thereof of the present invention. The hyperproliferative disease is preferably selected from disorders associated with, accompanied by or caused by AXL expression, overexpression or hyperactivity, such as cancer, e.g. breast cancer, colon cancer, lung cancer, kidney cancer, follicular lymphoma, myeloid leukemia, skin cancer/melanoma, glioblastoma, ovarian cancer, prostate cancer, pancreatic cancer, Barrett's esophagus and esophageal cancer, stomach cancer, bladder cancer, cervical cancer, liver cancer, thyroid cancer, and head and neck cancer, or hyperplastic and neoplastic diseases or other AXL expressing or overexpressing hyperproliferative diseases.
According to another preferred embodiment of the invention the cancer to be treated is a drug resistant cancer, preferably selected from the group of cancers cited above.
The invention further relates to a method of treating a disease wherein the antibody of the invention is administered to a mammal and wherein said disease is correlated directly or indirectly with the abnormal level of expression or activity of AXL.
Finally, the invention relates to a kit comprising an anti-AXL-antibody, preferably the antibody, antibody fragment or derivative thereof of the invention, the nucleic acid molecule encoding said components and/or the vector of the invention.
All embodiments covering the compounds disclosed herein can be used as single compounds or in combination for the preparation of a medicament.